High yield and rapid growth of Neoparamoeba pemaquidensis in co-culture with a rainbow trout gill-derived cell line RTgill-W1

Neoparamoeba pemaquidensis is an ubiquitous amphizoic marine protozoan and has been implicated as the causative agent for several diseases in marine organisms, most notably amoebic gill disease (AGD) in Atlantic salmon. Despite several reports on the pathology of AGD, relatively little is known about the protozoan and its relationship to host cells. In this study, an in vitro approach using monolayers of a rainbow trout gill cell line (RTgill-W1, ATCC CRL-2523) was used to rapidly grow large numbers of N. pemaquidensis (ATCC 50172) and investigate cell-pathogen interactions. Established cell lines derived from other tissues of rainbow trout and other fish species were also evaluated for amoeba growth support. The amoebae showed preference and highest yield when grown with RTgill-W1 over nine other tested fish cell lines. Amoeba yields could reach as high as 5 x 10(5) cells mL(-1) within 3 days of growth on the gill cell monolayers. The amoebae caused visible focal lesions in RTgill-W1 monolayers within 24 h of exposure and rapidly proliferated and spread with cytopathic effects destroying the neighbouring pavement-like cells within 48-72 h after initial exposure in media above 700 mOsm kg(-1). Disruption of the integrity of the gill cell monolayers could be noted within 30 min of exposure to the amoeba suspensions by changes in transepithelial resistance (TER) compared with control cell monolayers maintained in the exposure media. This was significantly different by 2 h (P < 0.05) compared with control cells and remained significantly different (P < 0.01) for the remaining 72 h that the TER was monitored. The RTgill-W1 cell line is thus a convenient model for growing N. pemaquidensis and for studying host-pathogen interactions in AGD.     

Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line

In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 degrees C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 degrees C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 degrees C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 degrees C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 degrees C) compared with the non-permissive temperature of 28 degrees C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis.     

暗纹东方鲀血细胞发生的观察

通过对暗纹东方鲀(Takifugu obscurus)血液涂片及头肾、体肾、脾脏和肝脏四种脏器印片的光镜观察,发现血细胞的发育大致经过三个阶段,即原始阶段、幼稚阶段、成熟阶段。实验对不同发育阶段的红血细胞、淋巴细胞、单核细胞、粒细胞进行了观察和测量,并对暗纹东方鲀血细胞的发生过程做了初步探讨。实验结果表明暗纹东方鲀血细胞的发育主要在头肾和体肾,肝脏印片未观察到原始造血细胞,提示肝脏可能不是暗纹东方鲀的造血器官。     

鲤鱼血淋巴细胞培养及染色体制备条件探索

从鲤鱼侧线鳞处静脉采血于含有肝素的一次性注射器中，采用RPMI1640全培养基进行体外淋巴细胞培养。通过对采血后细胞培养时间、秋水仙素浓度及加入时间、低渗时间及温度等条件的摸索，建立了较成熟的培养鱼类血淋巴细胞的实验方法。     

Culture of mantle epithelial cells expressing shell matrix proteins from scallop Patinopecten yessoensis

ABSTRACT:   In molluscs, mantle epithelial cells secrete organic matrix proteins to form shells. In this study, we established a culture of mantle epithelial cells by using the mantle pallial layer of scallops. We aimed to identify the mantle epithelial cells expressing scallop shell matrix proteins and establish a culture system of epithelial cells. After the mantle pallial layer was carefully isolated from the mantle tissue, explant culture was performed at 4°C. Most cells that migrated from the explant tissue were round cells. Most of the adhered cells retained round morphology, while some of the cells adhered to the dish and showed morphology similar to that of epithelial-like and fibroblast-like cells. When the cultured cells were immunostained with a polyclonal antibody against the shell matrix protein, the antibody recognized many of the adhered cells. An estimation of the number of epithelial cells revealed that approximately 70% of the adhered cells were epithelial cells. This is the first report to describe epithelial cells in cultured mantle cells, which express shell matrix proteins. This culture system may be a useful method for characterization of the mantle epithelial cells.     

松江鲈肾组织细胞系生物学特性研究

在25℃、5%CO_2实验室条件下,于20%FBS-DMEM/F12培养基中培养松江鲈肾组织细胞系,以研究其生物学特性。第75代松江鲈肾组织细胞传代培养后,0～2 d处于潜伏期,2～4 d进入对数生长期,4～6 d进入稳定期,其群体倍增时间为55.2 h。经液氮冷冻保藏1月后,复苏细胞的存活率为(90.8±1.37)%。35代松江鲈肾组织细胞的染色体数为2n=40,核型公式为K(2n)=16m+10sm+14t。病毒敏感性试验表明,松江鲈肾组织细胞对鱼类诺达病毒不敏感,对鲤春病毒血症病毒敏感,测定病毒滴度为10^6.53/mL。镉离子敏感性试验显示,松江鲈肾细胞存活率随着氯化镉浓度的升高而降低,半致死浓度为(130±9.1)μmol/L,可用来作为镉离子检测的生物学指标。     

锦鲤尾鳍细胞系(KF-H)的建立

本研究建立了锦鲤(Cyprinus carpio)尾鳍细胞系。染色体数目、核型及DNA含量等实验,发现锦鲤体细胞和锦鲤培养细胞无显著性差异,染色体数目和DNA含量符合比例关系,建立的锦鲤尾鳍细胞系已形成了稳定的遗传性状,命名为KF-H。     

Cell wall disruption: An effective strategy to improve the nutritive quality of microalgae in African catfish (Clarias gariepinus)

The rigid cell walls of microalgae may hinder their utilization in fish feeds. The current experiment assessed the correlation between the accessibility of microalgae nutrients and their in vivo digestibility in African catfish. Nannochloropsis gaditana biomass was subjected to physical or mechanical treatments to weaken its cell wall; untreated—no disruption treatment (UNT), pasteurization (PAS), freezing (FRO), freeze‐drying (FRD), cold pasteurization (L40) and bead milling (BEM). Six experimental diets formulated from differently treated and untreated microalgae (at 30% diet inclusion level) were tested on growth performance and apparent nutrient digestibility (ADCs) in juvenile African catfish. A basal diet (REF) containing no microalgae was used as reference diet. Results showed that biomass gain and feed conversion ratio of fish fed L40 and BEM diets increased by 13% and 11%, respectively, relative to the UNT diet. Additionally, FRD, FRO, L40 and BEM cell wall disruption treatments improved protein digestibility by 0.5%, 5.9%, 8.4% and 16.3%, respectively, compared to the UNT treatment. There was a positive correlation between accessibility of microalgal nutrients and their digestibility in African catfish. Nutrient digestibility of microalgae was dependent on extent of cell disruption. Also, the impact of cell disruption on nutrient digestibility of microalgae differs between African catfish and Nile tilapia.     

病毒感染过程中青鳉irf2基因过表达的双面性

为了探究干扰素调节因子2 (interferon regulatory factor,IRF2)如何通过调控干扰素(IFN)表达影响鱼类的免疫,实验从青鳉中克隆了irf2 (Olirf2),发现该基因在青鳉各个组织中均有表达;将构建的真核表达载体pTol2/CMV-IRF2/IE1-pr转染到胖头鱥肌肉细胞系(FHM)后,发现瞬时过表达Olirf2能够显著促进鲤春病毒血症病毒(spring viremia of carp virus,SVCV)的复制,并抑制抗病毒相关基因mx1、ifn和irf3的表达。进一步通过双荧光素报告系统发现,Olirf2能够显著抑制NF-κB和ISRE的活性,说明Olirf2可能通过抑制细胞的天然免疫应答进而促进病毒的增殖。然而持续过表达Olirf2则增强了细胞的抗病毒能力,同时促进干扰素相关基因mx1、ifn和irf3的表达。因此,Olirf2基于表达的持续时间不同而具有抗病毒或者促病毒的双面效果。实验通过研究Olirf2在抗病毒信号通路中发挥的作用,为通过基因编辑或者转基因手段来构建抗病毒的鱼类提供了理论基础。     

营养盐浓度对棕鞭藻叶绿素荧光参数、细胞密度和总脂含量的影响

利用生物化学方法和叶绿素荧光动力学分析技术,研究了棕鞭藻在一次性培养过程中,不同营养盐浓度(f/4、f/2、f、2f、4f)对其叶绿素荧光参数、叶绿素含量、细胞密度、单位体积干质量、总脂含量和总脂收获量的影响。试验结果表明,营养盐浓度为2f时,该藻的最终细胞密度、叶绿素含量、单位体积干质量、总脂含量和总脂收获量均显著高于其他各处理组,由此可以看出,在本试验条件下,最适合棕鞭藻生长和油脂积累的营养盐浓度是2f。不同营养盐浓度对棕鞭藻的叶绿素荧光参数有显著影响,培养6～10d,f/4处理组的叶绿素荧光参数(光系统Ⅱ的最大光能转换效率;光系统Ⅱ的潜在活性;光系统Ⅱ的实际光能转化效率;相对电子传递效率和光化学淬灭)最低,其次为f/2处理组(P0.05)。培养7～10d,2f处理组的光系统Ⅱ的实际光能转化效率、相对电子传递效率和光化学淬灭显著高于其他处理组(P0.05)。培养结束时(第10d),各处理组细胞密度和总脂含量依次为:2f4fff/2f/4。相关性分析结果表明,营养盐浓度为f/4～2f时,棕鞭藻的细胞密度、叶绿素含量、单位体积干质量和总脂含量与营养盐浓度均呈显著的正相关;营养盐浓度为2f～4f时,上述各值与营养盐浓度则均呈显著的负相关。